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PubMed1990

The use of immunogold-silver staining to study antigen variation and bacterial entry into eukaryotic cells by conventional light microscopy.

Immunogold-silver staining is a sensitive staining technique that enables the visualisation of the presence of individual antigens by conventional light microscopy. The application of this method to detect the antigenic heterogeneity of bacterial surface components and also the localisation of intracellular or extracellular bacteria is described. The latter application involved selective immuno-silver staining of the extracellular bacteria and counterstaining of the intracellular bacteria and the eukaryotic cells by crystal violet. The efficacy of the assay was confirmed by transmission electronmicroscopy of the silver-stained specimens. Immunogold-silver staining was shown to be useful for studying bacterial antigen variation and the uptake of bacteria by eukaryotic cells.

Antigenic Variation↗
PubMed1988

[Silver staining of the nucleoli of pig embryonic kidney cells during the hyperactivation and inhibition of the synthesis of nucleolar RNA by UV microirradiation].

Silver staining of the nucleoli in pig embryo kidney cells (PK) was studied during the cell cycle and also upon mature nucleoli modifications induced by UV microirradiation. During anaphase only four silver-stained granules were revealed in each daughter set of chromosomes in the four nucleolus-organizing regions (NORs). In the following 1-2 hours, the number of granules in the NORs rapidly increased up to 25-30 per nucleus. During the next 20-25 hours of the cell cycle, the number of silver-stained granules was slowly doubling as the nucleoli grew in size. UV microirradiation of one nucleolus in the nucleus with two nucleoli induced a profound degradation of the injured nucleolus and a compensatory hypertrophy of the intact one. Such nucleolar modifications were accompanied by redistribution of the silver-stained granules between the injured and non-injured nucleoli and by alterations in the levels of nucleolar RNA synthesis in the NORs. These data support a hypothesis that silver-stained proteins may be involved in the regulation of the nucleolar activity.

Animals↗
PubMed1989

An immunogold-silver staining method for detection of cell surface antigens in cell smears.

We developed an indirect immunogold-silver staining method for detection of leukocyte cell surface antigens in cell smears. Air-dried and fixed cytocentrifuge preparations or smears of peripheral blood leukocytes were incubated with monoclonal antibodies (MAb) and colloidal gold-labeled secondary antibodies. The preparations were post-fixed and silver enhancement was performed. The smears were counterstained with May-Grunwald-Giemsa and examined in brightfield light microscopy. The morphology of the cells was well preserved. Leukocytes reacting with the MAb showed black granules on their surface membranes. The intense immunostaining and the low background allowed a rapid enumeration of the positive cells. The labeling could be detected with high sensitivity by epipolarization microscopy. This immunogold-silver staining method was used to quantify T- and B-lymphocytes and natural killer cells in buffy coat smears of normal adult blood. These lymphocyte subsets correlated well with those obtained in smears with the alkaline phosphatase-anti-alkaline phosphatase (APAAP) method and with those found by labeling of mononuclear cells in suspension with immunogold-silver staining. This immunogold-silver staining method forms a good alternative to immunoenzyme methods for study of hematologic cells. In addition, it could be a general procedure for detection of cell surface antigens in all kinds of cell smears.

Antibodies, Monoclonal↗
PubMed1988

[Silver staining of nucleolus organizer regions (NORs). Application to the study of nucleolar structure and value in pathology].

Nucleolus is the morphologic expression of synthesis and maturation of ribosomal RNA (rRNA) from amplified ribosomal DNA (rDNA). Nucleolar Organizer Regions (NORs) are functional subunits of the nucleolus in which actively transcribed rDNA is surrounded with numerous regulatory proteins. Some of them are argyrophilic non-histone proteins (Ag-NOR proteins). By using a cytochemical reaction based on this argyrophilia, active NORs may be stained by the precipitation, at their level, of metallic silver granules whose quantity is directly related to the nucleolar activity. In the present paper, we described various applications of a silver-staining method we developed in our laboratory. The Ag-NOR proteins were ultrastructurally localized within precise nucleolar components. Moreover by viewing thick-sections of silver-stained cells with high-voltage microscopes we were able to describe the three-dimensional structure of nucleoli. Finally, this silver-staining method may be applied at the optical level, to sections of routinely fixed and paraffin-embedded human tissues. With this simple staining method, it is now possible to study the relationships of the number of nucleolar argyrophilic structures with the diagnostic of neoplasms.

Humans↗
PubMed1983

Morphological heterogeneity among Salmonella lipopolysaccharide chemotypes in silver-stained polyacrylamide gels.

The morphological heterogeneity of lipopolysaccharides (LPSs) among salmonella mutants with different LPS chemotypes was analyzed in silver-stained polyacrylamide gels. The biochemical differences in the LPS chemotypes were reflected in the unique profiles of the purified LPSs. The LPS profiles in the whole-cell lysates were also unique for each chemotype. (Whole-cell lysates were assessed by a method which preferentially silver stains LPS and by a proteinase K digest of whole-cell lysates. The silver-stained LPS profiles of proteinase K-digested lysates were similar to the homologous purified LPS and could be used to preliminarily characterize the LPS chemotype before purification.) In summary, biochemical variation in LPS composition can be detected in silver-stained polyacrylamide gels.

Electrophoresis, Polyacrylamide Gel↗
PubMed1991

A method for quantifying radioactivity associated with protein in silver-stained polyacrylamide gels.

A method is described in which individual proteins labeled with weak beta-emitting radionuclides, separated by polyacrylamide gel electrophoresis, and stained with silver are released from the gel by the use of the periodate soluble cross-linking agent N,N'-dialyltartardiamide. The radioactivity can then be quantitated using liquid scintillation counting. The method is shown to be insensitive to reasonable variations in the intensity of staining as well as the gel volume over a practical range of gel slices. Recovery from the gel is extremely good with 93% of the counts associated with 14C-labeled proteins of known radioactive concentration being recovered. Analysis of a complex mixture of 3H-labeled proteins indicates resolution similar to that obtainable by autoradiography without the problems associated with quenching of autoradiographic signal by the staining procedure. The method is used to determine the amount of fucose and mannose incorporated into a putative cell adhesion protein during development of the cellular slime mold Dictyostelium purpureum.

Autoradiography↗
PubMed1985

Oligoclonal bands in unconcentrated CSF. Silver stain for agarose gels.

Agarose gel electrophoresis (AGE) and agarose isoelectric focusing (AIEF) were performed on unconcentrated CSF from patients with multiple sclerosis and other neurological disorders. Electrophoretic profiles were examined after staining with silver. Oligoclonal distribution of immunoglobulins were observed in over 90% of patients with clinically definite multiple sclerosis and over half of all patients with possible or probable multiple sclerosis. Up to 20 samples could be simultaneously examined and required the use of only 2-20 microliter of unconcentrated CSF containing 10-100 ng of total IgG. The entire procedure from sample application to completion of staining required less than 3 h. The techniques offered exquisite resolution of abnormal immunoglobulin bands and permitted distinction of normal from abnormal CSF readily.

Electrophoresis, Agar Gel↗
PubMed1990

Silver stains for identification of neuroendocrine cells. A study of the chemical background.

The chemical background of silver stains used for visualization and characterization of peripheral neuroendocrine cells in the gastrointestinal tract and pancreas, and of their corresponding tumours, was studied in tissue sections and by a dot-blot technique. Sequential staining of pancreatic islets with an immunohistochemical procedure and silver staining of the same tissue section revealed that chromogranin A immunostained cells also displayed an argyrophil reaction with the Grimelius method, but no argentaffin reaction with the Masson technique. Accordingly, purified chromogranin A (15 micrograms or less) treated in formalin and applied to nitrocellulose did not show any argentaffin reaction but displayed a dose-related argyrophil reaction. Equal quantities of other polypeptide components did not give rise to any silver reaction. Further dot-blot studies showed that the tryptophan and tyrosine metabolites, dopamine, norepinephrine, 5-hydroxytryptamine and 5-hydroxinodole caused strongly argentaffin and argyrophil reactions while epinephrine, 5-hydroxyindole-3-acetic acid and 5-hydroxytryptophan gave only the former reaction. Among other chemical components studied, only guanine displayed weak silver staining. The results indicate that the reaction products between aldehydes and the granular content of biogenic amines synthesized from tryptophan and tyrosine display an argentaffin reaction and that the granular chromogranin A caused an argyrophil but no argentaffin reaction.

Animals↗
PubMed1980

Silver-stained synaptonemal complexes of human pachytene bivalents studied by light microscopy.

The synaptonemal complex (SC), a part of the ultrastructure of the pachytene bivalent of eukaryotic organisms, is intimately connected with the pairing of homologous chromosomes. Its development, structure, and function have been studied extensively with the electron microscope during the past 20 years. A simple method of staining with silver nitrate has made it possible for us to visualize human SCs with the light microscope.

Cell Cycle↗
PubMed1986

Detection of oligoclonal IgG bands in unconcentrated CSF by isoelectric focusing in agarose gel and silver staining.

We analyzed 102 unconcentrated cerebrospinal fluids from a variety of neurologic diseases for oligoclonal IgG bands by isoelectric focusing in agarose gel followed by staining with silver. Ten to 12 microliter of cerebrospinal fluid containing 0.4-0.8 microgram of IgG was found to be optimum. Cerebrospinal fluid from 38 of 40 patients with clinically definite multiple sclerosis, 6 of 9 with suspected multiple sclerosis and 8 of 53 patients with other neurologic diseases had oligoclonal IgG bands by IEF in agarose followed by immunofixation. The commercial system employed here is a simple sensitive and rapid method for detection of oligoclonal bands in unconcentrated cerebrospinal fluid of patients with multiple sclerosis.

Electrophoresis, Agar Gel↗
PubMed1996

Cloning and mapping of variety-specific rice genomic DNA sequences: amplified fragment length polymorphisms (AFLP) from silver-stained polyacrylamide gels.

An efficient technique for cloning DNA from silver-stained denaturing polyacrylamide gels was developed to allow the isolation of specific bands obtained from selective restriction fragment amplification (SRFA). This method proved as reliable as cloning radioactively labelled SRFA bands from the same gels. Rice DNA was used as a template, both with and without [32P]dCTP, using the same PCR profiles. Amplified products were separated using denaturing polyacryamide gel electrophoresis and visualized either by silver staining of gels or by autoradiography of 32P-labelled products. We cloned specific polymorphic SRFA bands directly from the denaturing polyacrylamide gels with one round of PCR amplification and confirmed that the sequences of the bands from silver-stained gels were identical to the corresponding 32P-labelled bands. The bands that were chosen represented amplified fragment length polymorphisms (AFLPs) between japonica and indica rice varieties. We studied the ability of two cloned AFLP bands to serve as heritable genetic markers by mapping them as RFLPs in an interspecific rice population and found that they represented single-copy DNA at unique loci in the rice genome.

Base Sequence↗
PubMed2002

Fully reversible procedure for silver staining improves densitometry of complex mixtures of biopolymers resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Due to its high sensitivity, silver staining is a widely popular method for the revelation of biopolymers separated by both native and denaturing electrophoresis. A step-by-step method for the destaining and restaining of overdeveloped/overloaded silver-stained bands is described that is applicable to both proteins and nucleic acids. The procedure significantly improves densitometric analysis of gels that have been silver stained with either commercial kits or solutions made in-house. The method permits reproducible densitometry of silver-stained gels and allows quantification of both main and minor components in complex mixture of molecules resolved on the same gel slab. All steps may be interrupted and are readily reversible, allowing for facile densitometric analyses and photographic recording under optimized conditions. Furthermore, common artifacts such as differential staining of the two gel surfaces, localized uneven yellow-ochre background, and the presence of fold marks and fingerprints can be easily removed.

Artifacts↗
PubMed1987

The effect of pH on silver staining of nucleolus organizer regions.

The effect of pH on silver staining of the nucleolus organizer regions (NORs) of human chromosomes has been investigated between pH 6.5 and 12.0. Nonvolatile mixtures of ethanolamine and ethanolammonium nitrate replaced the ammonia of standard procedures. The optimal NOR staining obtained at pH 3.5 by the silver staining procedure of Howell and Black served as a standard; this procedure stained all ten NORs in 90% of mitoses. Similar NOR staining was found in 75% of mitoses stained at pH 11.7 or 11.8, but only in 10-15% of mitoses stained between pH 11.6 and 10.0. Between pH 10.0 and 9.0 NOR staining was incomplete, and between pH 8.5 and 6.5 there was no NOR staining.

Chromosome Banding↗
PubMed1991

Transfer of silver-stained proteins from polyacrylamide gels to polyvinylidene difluoride membranes.

We have developed a method to transfer proteins from a silver-stained polyacrylamide gel to a polyvinylidene difluoride (Immobilon-P) transfer membrane (Millipore, Bedford, MA). If the silver stained gels are rinsed in 2 x SDS Laemmli sample buffer prior to transfer, almost all proteins can be transferred comparably to non-stained controls. Some proteins stained with silver can be directly transfer, almost all proteins can be transferred comparably to non-stained controls. Some proteins stained with silver can be directly transferred to a single sheet of Immobilon-P without a prior rinse in sample buffer. Most important in the Western blot the antigenicity of the transferred protein is retained in either way. The method described is simple, inexpensive and versatile. A slight modification of the technique permits one to extract minor proteins, or detect their antigenic activities, without contamination of contiguous proteins.

Blotting, Western↗
PubMed1985

Comparison of detection of oligoclonal immunoglobulin G bands in cerebrospinal fluid specimens by Coomassie blue and silver stains.

Cerebrospinal fluid (CSF) specimens from 45 clinically definite MS patients were studied for the presence of oligoclonal IgG bands by sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The protein bands were identified by Coomassie blue (CB) dye and silver staining. Thirteen patients showed no bands in their CSF by the CB method. Nine of these 13 patients showed presence of bands by silver staining alone. Thirty-two patients showed more than two bands in their CSF by either technique, and 28 of the 32 patients showed from one to five additional bands by silver staining compared to Coomassie blue staining. The silver staining method was thus more sensitive in identification of oligoclonal bands in CSF.

Clone Cells↗
PubMedDate not supplied

[Demonstration of viral proteins by silver staining].

Investigations were conducted to establish the optimal conditions for silver staining of polypeptides separated by polyacryle-amide gel electrophoresis. The thickness of the gel layer was of 1.5 mm. The experiments showed that the modified Hankeshover and Dernick's technique (4) (replacement of the fixative for the electrophoretically separated polypeptides and extension of the bleaching by Farmer's reagent time) had the best sensitivity and reproducibility. This technique can be performed also on polyacryle-amide gel preparations already stained by the Coomassie Brilliant Blue method. To check the efficiency of this silver staining technique, the authors used it to detect the Sendai virus and B hepatitis virus surface antigen polypeptides.

Electrophoresis, Polyacrylamide Gel↗
PubMed1991

Alcian blue fixation allows silver staining of the isolated polysaccharide component of bacterial lipopolysaccharides in polyacrylamide gels.

The effect of the cationic dye Alcian Blue on the silver staining of bacterial lipopolysaccharide and its polysaccharide and lipid A portions in polyacrylamide gels was investigated. The polysaccharide was only stained when the gel was previously treated with the dye. The polysaccharide moiety was found to be responsible for the lipopolysaccharide staining with silver, whereas the lipid A seemed unimportant. Treatment with Alcian Blue may prove useful to detect hydrophilic components of lipopolysaccharide samples that could not be stained by the usual silver staining procedures.

Alcian Blue↗
PubMed1984

Silver stain for proteins on a cellulose acetate membrane.

A rapid and sensitive silver staining method to detect proteins on a cellulose acetate membrane has been established. This method is achieved by modification of the silver-based color staining for detection of proteins in polyacrylamide gels [D. W. Sammons, L. D. Adams, and E. E. Nishizawa, Electrophoresis 2, 135-141 (1981)] and applied to our new type of two-dimensional electrophoresis for analysis of proteins on a cellulose acetate sheet [T. Toda, T. Fujita, and M. Ohashi, Anal. Biochem. 119, 167-176 (1982)]. Maximal sensitivity of silver stain for proteins on a cellulose acetate membrane can be obtained by an optimal balance between deposition of silver on the protein and on the background. Certain kinds of proteins are colored red, orange, or grayish-blue. The silver stain is 20-80 times more sensitive than Coomassie blue and some spots are visualized reproducibly by silver only. Densitometric evaluation of standard proteins stained with silver and Coomassie blue is also demonstrated. The method takes only 50 min to perform and is sensitive, simple, and reproducible.

Animals↗